Novel mycobacterium tuberculosis extracellular nuclease and application thereof

A Mycobacterium tuberculosis, exonuclease technology, applied in applications, hydrolase, microorganism-based methods, etc., can solve problems such as no data showing Mycobacterium tuberculosis

Active Publication Date: 2016-03-23
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although M. tuberculosis intracellular nucleases have been described in previous reports, to date there are no data showing the presence of M. tuberculosis extracellular nucleases

Method used

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  • Novel mycobacterium tuberculosis extracellular nuclease and application thereof
  • Novel mycobacterium tuberculosis extracellular nuclease and application thereof
  • Novel mycobacterium tuberculosis extracellular nuclease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning and effect verification of Mycobacterium tuberculosis extracellular nuclease Rv0888

[0039] 1. Materials and methods

[0040] 1.1 Animals, strains and growth conditions

[0041] Mice were purchased from Beijing Weitong Lihua. All animals used in the experiment followed the animal welfare and ethics of Heilongjiang Animal Ethics Committee of Heilongjiang Science and Technology Department of the People's Republic of China, and were approved and supervised by the Animal Welfare Committee of Harbin Veterinary Research Institute. All experiments used the fewest number of mice possible and minimized animal suffering.

[0042] Mycobacterium smegmatis (M.smegmatis) was cultured in Middlebrook 7H9 (BD Biosciences) containing 10% OADC (oleicacid / albumin / catalaseen richment, BD Biosciences), 0.05% Tween-80 (Amresco), and 0.2% glycerol (Sigma-Aldrich) . E.coliDH5α strain was used for plasmid preparation, and E.coliRosetta2 strain was used for protein expressio...

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Abstract

The invention discloses novel mycobacterium tuberculosis extracellular nuclease and application of the novel mycobacterium tuberculosis extracellular nuclease, and proves that Rv (Rotavirus) 0888 is extracellular nuclease of mycobacterium tuberculosis. Based on a sequence analysis, no homology exists between the Rv0888 nuclease and known extracellular nuclease, so that the Rv0888 is proved to be novel extracellular nuclease; activity exerting of Rv0888 protein needs the assistance of divalent ions, and the optimal temperature and the optimal pH (Potential of Hydrogen) of the Rv0888 protein are respective 41 DEG C and 6.5. The invention also provides application of four kinds of traditional Chinese medicine monomers of oleuropein, 6-gingerol, corylifolinin and acteoside for inhibiting the novel mycobacterium tuberculosis extracellular nuclease. Site-directed mutagenesis research shows that a catalytic action of H353 residue, D387 residue and D438 residue on the activity of the Rv0888 is achieved; an in-vivo infection experiment proves that the persistence capacity of recombined mycobacterium smegmatis Rv0888 NS/MS and Rv0888 S/MS in a lung of a mouse is obviously higher than that of pMV262/MS, obvious pathological change can be generated in the lung of the mouse by the Rv0888 NS/MS and the Rv0888 S/MS, and the Rv0888 is proved to be necessary for mycobacterium infection and pathogenicity.

Description

technical field [0001] The invention relates to an extracellular nuclease and its use, in particular to a novel Mycobacterium tuberculosis extracellular nuclease and its use. The invention belongs to the field of biotechnology. Background technique [0002] Many species of bacteria produce extracellular nucleases, which play an important role in bacterial virulence, biofilm formation, utilization of extracellular DNA for nutrients, and degradation of DNA in extracellular traps (NETs). For example, the extracellular nucleases of Shewanella, Serratia, and Staphylococcus aureus can degrade extracellular DNA to obtain their basic nutrients-phosphorus, carbon and nitrogen sources. Seper's recent study showed that wild-type Vibrio cholerae can degrade DNA components in NETs through its own two extracellular nucleases (Dns and Xds). S. aureus ectonucleases also degrade NETs, ​​triggering caspase-3-mediated immune cell death. However the ectonuclease (Gbs0661) of S. agalactiae is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12R1/32
CPCC12N9/22
Inventor 刘思国陈利苹宋宁宁
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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